The ever-increasing global demand for energy has led to depletion of fossil fuels, which are buried combustible geologic deposits of organic materials that have been converted to crude oil, coal, natural gas, or heavy oils. Because fossil fuels are formed by exposure to heat and pressure in the earth's crust over hundreds of millions of years, they are a finite, non-renewable resource. Accordingly, there is a need for non-fossil fuel energy sources.
Hydrocarbons from biological sources represent a clean, sustainable alternative energy source. Further, many industries, including plastics and chemical manufacturers, rely heavily on the availability of hydrocarbons for manufacturing processes. Currently, energy-rich lipids and fatty acids (“nature's petroleum”) are isolated from plant and animal oils to produce diverse products such as fuels and oleochemicals. Recent efforts have focused on the microbial production of fatty acids and fatty acid derivatives by cost-effective bioprocesses. Methods of producing fatty acids and/or fatty acid derivatives in microbial hosts are described in, e.g., PCT Publication Nos. WO 2007/136762, WO 2008/119082, WO 2009/009391, WO 2009/076559, WO 2009/111513, WO 2010/006312, WO 2010/044960, WO 2010/118410, WO 2010/126891, WO 2011/008535, and WO 2011/019858, and in Schirmer et al., Science 329(5991):559-562 (2010).
Long chain fatty alcohols possess high energy density relative to shorter-chain biofuel products such as ethanol, and can be produced in cultured cells via a series of enzymatic processes. Fatty alcohols can also be enzymatically or catalytically converted to hydrocarbons such as alkenes. Fatty alcohols and their derivatives have numerous commercial applications, including use as surfactants, lubricants, plasticizers, solvents, emulsifiers, emollients, thickeners, flavors, fragrances and fuels.
Enzymes that convert fatty acyl-CoA to fatty alcohols or fatty aldehydes are commonly known as fatty acyl-CoA reductases (“FARs”). FARs have been identified in, e.g., Euglena (see, e.g., Teerawanichpan et al., Lipids 45:263-273 (2010)), Arabidopsis (see, e.g., Rowland et al., Plant Physiol. 142:866-877 (2006), Doan et al., J. Plant Physiol. 166:787-796 (2009) and Domergue et al., Plant Physiol. 153:1539-1554 (2010)), Artemisia (see, e.g., Maes et al., New Phytol. 189:176-189 (2011)), jojoba (see, e.g., Metz et al., Plant Physiol. 122:635-644 (2000)), moth (see, e.g., Lienard et al., Proc. Natl. Acad. Sci. 107:10955-10960 (2010)), bee (see, e.g., Teerawanichpan et al., Insect Biochemistry and Molecular Biology 40:641-649 (2010)) and in mammals (see, e.g., Honsho et al., J. Biol. Chem. 285:8537-8542 (2010)). Alcohol-forming acyl-CoA reductases are thought to generate fatty alcohols directly from acyl-CoA. Enzyme-based conversion of acyl-CoA to fatty alcohol can also occur in a two-enzyme, two-step reaction; in the first step, acyl-CoA is reduced to fatty aldehyde by an aldehyde-forming acyl-CoA reductase, and in the second step, the fatty aldehyde is reduced to a fatty alcohol by a fatty aldehyde reductase.
Typically, to produce fatty alcohols in a microorganism, it is necessary to introduce various enzymes in addition to at least one FAR. For example, in a host that does not endogenously produce acyl-CoA, such as a cyanobacterial host, it may be necessary to introduce, e.g., a fatty acid thioesterase to convert acyl-acyl carrier protein (acyl-ACP) to free fatty acids and acyl-CoA synthetase to convert free fatty acids to acyl-CoA, in addition to an acyl-CoA reductase to reduce acyl-CoA to fatty alcohols. Even in host organisms that naturally produce acyl-CoA, it can be advantageous to introduce a gene encoding an acyl-CoA synthetase (and, in many cases, an acyl-ACP thioesterase or an acyl-CoA thioesterase to provide the fatty acid substrate for the acyl-CoA synthetase) such that acyl-CoA is produced in higher amounts than occur in the absence of acyl-CoA synthetase overexpression. Other enzymes that can be engineered into a host strain to provide substrates for alcohol-forming FARs include aldehyde-forming acyl reductases such as aldehyde-forming acyl-CoA reductases (e.g., Reiser amd Somerville (1997) J. Bacteriol. 179: 2969-2975; Wang and Kolattukudy (1995) FEBS Lett. 370: 15-18; Vioque and Kolattukudy (1997) Arch. Biochem. Biophys. 340: 64-72) or aldehyde-forming acyl-ACP reductases (e.g., WO 2011/006137). Introducing several heterologous pathway components, however, may lead to difficulties in appropriately balancing enzyme expression and activity to produce the desired end product in sufficiently high yields for large scale production. Moreover, the buildup of intermediates such as free fatty acids can be toxic to the host cell, further reducing yield.
Secretion of a free fatty acid or a fatty acid derivative such as a fatty alcohol can also improve the efficiency of a production system. The multidrug resistance (MDR) transporters of prokaryotes can export a wide variety of substrates using energy supplied by either hydrolysis of ATP or a proton or sodium-motive force. The ATP-binding cassette (ABC) “primary transporters” or efflux pumps use ATP hydrolysis to drive substrate export, whereas the “secondary transporters” use proton motive force or proton motive force to drive substrate export. Included in the family of secondary transporters or efflux pumps are the Major Facilitator Superfamily (MFS), the Small Multidrug Resistance (SMR) family, the Resistance-cell Division (RND) family, the Multi Antimicrobial Extrusion (MATE) family, and the Putative E Transporter (PET) family (Mazurkiewicz et al. (2005) Cur Issues Mol Biol 7: 7-22; Haley and Saier (2000) J. Mol. Microbiol. Biotechnol. 2: 195-198). Prokaryotes also have protein secretion systems, currently classified as Secretion Systems I to VI, that transport effector proteins out of the bacterial cell (Shrivastava and Mande (2009) PLoS ONE 3: e2955).